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Published ahead of print on November 14, 2003, doi:10.1164/rccm.200305-669OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 454-458, (2004)
© 2004 American Thoracic Society

Mouse Lysozyme M Is Important in Pulmonary Host Defense against Klebsiella pneumoniae Infection

Philipp Markart, Thomas R. Korfhagen, Timothy E. Weaver and Henry T. Akinbi

Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio

Correspondence and requests for reprints should be addressed to Henry T. Akinbi, M.D., Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229–3039. E-mail: henry.akinbi{at}cchmc.org


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Klebsiella pneumoniae is a common virulent causative agent for pneumonia. Lysozyme has previously been shown to play an important role in nonimmune host defense of the airways. This study was undertaken to assess the role of lysozyme M, the major isoform of lysozyme in mouse lung, in the killing of K. pneumoniae in lysozyme M-/- mice and transgenic mice with increased expression of lysozyme (lysozymetg mice). The airways of lysozyme M-/- mice maintained in a pathogen-free facility were colonized by Lactobacilli, a component of the oropharyngeal flora. No lactobacilli were detected in the lungs of wild-type (WT) or lysozymetg mice. Twenty-four hours after intratracheal infection with K. pneumoniae, bacterial killing was enhanced 9-fold in lysozymetg mice compared with WT mice and 43-fold compared with lysozyme M-/- mice. In survival studies, no lysozyme M-/- mice survived beyond 72 hours after infection, whereas 75% of lysozymetg (p < 0.01) and 25% of WT mice survived to 120 hours (p < 0.01). Deficiency of lysozyme M in the lungs increased susceptibility to K. pneumoniae infection, whereas increased expression of lysozyme conferred resistance to infection and enhanced survival.

Key Words: lysozyme • knockout • Klebsiella pneumonia

Lysozyme is a widely distributed bacteriolytic enzyme that hydrolyzes the ß-1,4 glycosidic bond between N-acetyl muramic acid and N-acetyl glucosamine in the cell wall of bacteria. Two lysozyme genes are expressed in the mouse (1, 2): Lysozyme P mRNA is expressed at high levels in small intestine (Paneth cells) and at much lower or undetectable levels in other cells/tissues; mRNA-encoding lysozyme M is predominantly expressed in macrophages, bone marrow, and lung tissue and is detected primarily at lower levels in other tissues. In lung tissues, lysozyme protein was detected in alveolar macrophages, alveolar type II epithelial cells, and bronchoalveolar lavage fluid (BALF) (3–5). Consistent with the results of previous S1 nuclease studies (2), lysozyme M was recently identified as the major isoform of the enzyme in BALF (6).

Lysozyme is one of the most abundant antimicrobial proteins in the airways. The concentration of the enzyme in human airway surface liquid is estimated to be 20–100 µg/ml, sufficient to kill two important pulmonary pathogens: Staphylococcus aureus and Pseudomonas aeruginosa (7). Targeted expression of the rat homologue of mouse lysozyme M in distal respiratory epithelial cells of transgenic mice significantly increased killing of P. aeruginosa and group B Streptococcus, leading to enhanced survival of transgenic mice (3). The results of these studies indicate that lysozyme M plays an important role in nonimmune host defense of the airways and suggest that deficiency of this enzyme may increase susceptibility to infection.

Klebsiella pneumoniae is a frequent cause of nosocomial infections and may be responsible for as much as 20% of respiratory infections in the neonatal intensive care unit (8). The emergence of multidrug resistant strains of K. pneumoniae has significantly complicated the management and treatment of infections involving this organism. This study was therefore undertaken to assess the role of lysozyme M in protecting the airways of mice against infection by K. pneumoniae. The importance of lysozyme M for killing of K. pneumoniae in vivo was assessed by genetic manipulation of lysozyme concentration in the airspaces of transgenic mice.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Three groups of mice, lysozyme-overexpressing transgenic mice (lysozymetg), lysozyme M-deficient mice (lysozyme M-/-), and strain/age-matched wild-type (WT) mice, were used in these studies. Lysozymetg mice (FVB/N strain) were generated by targeting expression of the rat lysozyme cDNA to the distal respiratory epithelium under the direction of the 3.7-kb human surfactant protein-C (SP-C) promoter, as previously reported (3). Muramidase activity was increased 16-fold in the BALF of transgenic mice relative to WT mice. Lysozyme M-/- mice were generated in the C57BL/6 background by insertion of the gene encoding enhanced green fluorescent protein into the lys locus, as previously reported (9). Lysozyme M-/- mice used in this study were backcrossed 10 generations with FVB/N mice to facilitate comparison with lysozymetg mice. All mice were housed in a pathogen-free barrier facility in which sentinel mice were periodically monitored for common murine pathogens. For each experiment, 5- to 6-week old lysozymetg and lysozyme M-/- were compared with WT FVB/N mice. All mice were handled according to the institutional animal care and use committee guidelines at Cincinnati Children's Hospital Medical Center.

Bacteria
K. pneumoniae of the KPA1(cps K2) serotype (10) used in this study is heavily encapsulated and is particularly virulent in mice. To minimize variability in virulence, bacteria used in all studies were from the same passage that had been frozen at -70°C in 16% glycerol/trypticase soy broth (trypticase soy broth). Preparation of bacteria for intratracheal injection is as previously reported (3) and as detailed in the online supplement. For each experiment, the dose was confirmed by plating an aliquot from the bacterial suspension that was injected.

Bacterial killing.
Preliminary experiments identified a dose of intratracheal K. pneumoniae that caused substantial inflammation with minimal mortality in WT FVB/N mice. One thousand CFUs of K. pneumoniae suspended in 100 µl of sterile phosphate-buffered saline (PBS) were administered intratracheally as described previously (3) and as detailed in the online supplement. Mice were sacrificed at 6, 24, or 72 hours after infection and lung homogenates plated on LB agar. Systemic dissemination was assessed by plating splenic homogenates on LB plates. Infection studies were performed at least three times. Data were pooled and expressed as mean CFU ± SEM per g of lung.

Survival studies.
Five- to six-week-old lysozyme M-/-, lysozymetg, and strain-matched WT mice (n = 20–25 for each group) were intratracheally infected with 105 CFU of K. pneumoniae suspended in 100 ml of sterile PBS. Water and food were provided ad libitum during the period of observation. The number of surviving mice was counted every 12 hours up to 120 hours, at which time all mice were sacrificed.

Cytokine levels in BALFs and lung homogenates.
Five 5- to 6-week-old mice from each genotype and WT mice were treated with PBS or 104 CFUs of K. pneumoniae by intratracheal administration. Twenty-four hours after treatment, levels of cytokines associated with severe lung injury (mouse interleukin [IL]-10, IL-12, IL-6, tumor necrosis factor-{alpha}, macrophage inflammatory protein-2, and released upon activation normal T cell expressed and secreted) were assessed in BALF (uninfected mice) or lung homogenates (K. pneumoniae-infected mice) as previously described (3) and as detailed in the online supplement. All samples were assayed in duplicate.

BALF cell count.
Total and differential cell counts were assessed on BALF obtained from five 5- to 6-week-old lysozymetg, lysozyme M-/-, and WT control mice 24 and 72 hours after intratracheal infection with K. pneumoniae, as detailed in the online supplement.

Assessment of lung sterility.
Ten 5- to 6-week-old lysozyme M-/-, lysozymetg, or WT mice, housed in a pathogen-free barrier facility, were sacrificed and the sterility of the lungs assessed by plating lung homogenates on blood agar and incubating overnight at 37°C. Bacteria were Gram stained to assess the morphology and were further characterized for catalase activity, as detailed in the online supplement.

Lung Histology
Lung morphology was assessed on paraffin-embedded sections from five 5- to 6-week-old transgenic lysozymetg, lysozyme M-/-, or WT mice 24 or 72 hours after intratracheal instillation of 104 CFU of K. pneumoniae or PBS (24 hours), as previously described (11). Lung sections from each lobe were stained with hematoxylin and eosin or immunoreacted with anti–matrix metalloproteinase-9 at 1:2,000 dilution so that only neutrophils were stained, as detailed in the online supplement.

Statistical Analysis
Data are expressed as mean ± SEM. For bacterial clearance, data are reported as absolute counts per gram of lung tissue. Differences between groups were analyzed by one-way analysis of variance, and differences between means were assessed by contrast comparisons and the Student-Newman-Keuls test (Statview; SAS Institute, Cary, NC). Nonparametric survival distribution was estimated to examine the differences in survival among groups and was subsequently analyzed using Kaplan-Meier curve statistics.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Assessment of Lung Sterility in Uninfected Lysozyme M-/- and Lysozymetg Mice
The role of lysozyme M in maintaining the sterility of the lungs was assessed in uninfected lysozyme M-/-, lysozymetg, and WT mice housed in a pathogen-free environment. One hundred to 5,000 CFU of bacteria were detected in cultures of lung homogenates plated on sheep blood trypticase soy agar from all 10 lysozyme M-/- mice, whereas no bacteria were recovered from the lung homogenates of 10 WT littermates or lysozymetg mice. Bacteria in lung homogenates from lysozyme M-/- mice were identified as lactobacilli based on the morphology of Gram-stained bacteria and a negative catalase reaction. To determine whether the presence of lactobacilli in the distal airway was associated with lung inflammation, BALFs from lysozyme M-/- mice were assessed for markers of inflammation. Total and differential cell counts and levels of proinflammatory (tumor necrosis factor-{alpha}, IL-1, IL-6, macrophage inflammatory protein-2) and antiinflammatory (IL-10) mediators were not significantly different among lysozyme M-/-, lysozymetg, and WT mice (data not shown). Evidence of an inflammatory reaction was also not apparent on lung sections from uninfected mice. These data indicate the distal airways were colonized without provoking an inflammatory response.

Killing of K. pneumoniae in Lysozyme M-/- and Lysozymetg Mice
Lysozymetg, lysozyme M-/-, and WT control mice were infected intratracheally with K. pneumoniae. Bacterial burden was not significantly different among the three genotypes at 6 hours after infection (Figure 1) . By 24 hours after infection, lung bacterial burden was decreased ninefold in lysozymetg mice and increased fivefold in lysozyme M-/- mice relative to WT mice (p < 0.01). Bacterial burden was increased 43-fold in lysozyme M-/- mice compared with lysozymetg mice (p < 0.01). By 72 hours after infection, 25% of lysozyme M-/- mice had died, whereas all WT and lysozymetg mice were alive. Bacterial burden was significantly increased in surviving M-/- mice compared with WT and lysozymetg mice (p < 0.01) (Figure 1). Sixty percent of lysozymetg mice had completely cleared their lungs of K. pneumoniae at 72 hours after infection. There was no background lactobacilli growth when lung homogenates were plated on Luria-Bertani plates at the dilutions used for quantitative cultures. K. pneumoniae was only occasionally recovered from splenic homogenates from lysozyme M-/-, indicating that bacterial infection was localized mainly to the lungs. Histologic examination of lung sections showed diffuse lobar consolidation, especially of the right lung in all three groups of animals at 24 hours after infection. By 72 hours, lungs from M-/- mice remained consolidated with neutrophil infiltrates, whereas there was virtually complete resolution of pneumonia in WT and lysozymetg mice (Figures 2A and 2B) . No pathology was detected in the conducting airways.



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  		Figure 1. Lung bacterial burden after intratracheal instillation of K. pneumoniae. Lysozyme M-/- (black bars), lysozymetg (hatched bars), and wild-type (WT) (white bars) mice were infected with 104 CFUs of K. pneumoniae. Lungs were harvested, weighed, and homogenized at indicated time points. The numbers of viable bacteria were assessed by colony counting and expressed as CFUs/g lung tissue. Fifteen to 22 mice of each genotype were assessed at each time point. *p < 0.01 WT versus lysozymetg and **p < 0.01 for WT versus lysozyme M-/- mice.

 



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  		Figure 2. (A) Representative lung histology after intratracheal instillation of phosphate-buffered saline (PBS) (24 hours) or 104 CFU of K. pneumoniae (24 and 72 hours). Sections from five lysozyme M-/-, lysozymetg, or WT mice were stained with hematoxylin and eosin at the indicated time points. Scale bar = 50 µm. (B) Neutrophilic infiltration at 72 hours after infection. Tissue sections from lysozyme M-/- mice at 24 and 72 hours after infection were stained with antibody directed against matrix metalloproteinase-9 at a dilution (1:2,000) that specifically detected neutrophils. Scale bar = 50µm.

 
Survival of Lysozyme M-/- and Lysozymetg Mice after K. pneumoniae Infection
Survival studies were undertaken to assess the incidence of mortality after intratracheal infection with a larger dose of K. pneumoniae (Figure 3) . All mice survived the first 36 hours, although most of the mice were in respiratory distress. The mice appeared lethargic and fed poorly. By 72 hours after infection, all of the lysozyme M-/- mice had succumbed, whereas 25% of WT mice and 75% of the lysozymetg mice survived (p < 0.04 WT vs. lysozyme M-/-, p < 0.01 lysozymetg vs. WT). Survival was unchanged for WT and lysozymetg mice from 72 to 120 hours after infection.



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  		Figure 3. Survival after intratracheal instillation of K. pneumoniae. Lysozyme M-/-, lysozymetg, or WT mice (n = 20–25 for each genotype) were infected by intratracheal instillation of 105 CFU of K. pneumoniae. The number of surviving mice was documented until 120 hours after infection. *p < 0.04 WT versus lysozyme M-/- mice; **p < 0.01 WT versus lysozymetg mice.

 
Inflammation in Lysozyme M-/- and Lysozymetg Mice after K. pneumoniae Infection
There were no significant differences in the total cell counts and percentage neutrophils in BALFs from uninfected lysozyme M-/-, lysozymetg, and WT mice (data not shown). Both total cell counts and percentage neutrophils were significantly increased in BALF 24 hours after infection with K. pneumoniae; however, there were no significant differences in total or differential counts among the three genotypes at this time point. Cell counts remained significantly elevated in all three genotypes up to 72 hours after infection, but the percentage of neutrophils in BALF from lysozyme M-/- mice was increased 2.4-fold relative to WT mice and 4.3-fold relative to lysozymetg mice (Figure 4) .



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  		Figure 4. Total cell count (A) and percentage neutrophil count (B) in bronchoalveolar lavage fluid (BALF) after intratracheal instillation of either PBS or K. pneumoniae. Lysozyme M-/- (black bars), lysozymetg (hatched bars), and WT (white bars) mice were infected with 104 CFU of K. pneumoniae or instilled with PBS. Five mice were assessed for each genotype at each time point. *p < 0.01 for lysozyme M-/- compared with WT and lysozymetg mice.

 
To determine whether the inflammatory response to K. pneumoniae infection was modulated by the gain or loss of lysozyme, the concentration of selected inflammatory mediators was assessed in lung homogenates from lysozyme M-/-, lysozymetg, and WT mice. Levels of IL-6, IL-10, IL-12, tumor necrosis factor-{alpha}, macrophage inflammatory protein-2, and released upon activation normal T cell expressed and secreted were assessed 24 hours after infection. Relative to uninfected mice, the proinflammatory cytokines tumor necrosis factor-{alpha}, IL-6, macrophage inflammatory protein-2, and released upon activation normal T cell expressed and secreted were significantly increased in all three groups of mice after intratracheal infection with K. pneumoniae (Figure 5) . Although tumor necrosis factor-{alpha}, IL-6, macrophage inflammatory protein-2, and released upon activation normal T cell expressed and secreted did not differ among the three groups, the concentration of the antiinflammatory cytokine IL-10 was significantly decreased in lysozymetg mice and significantly increased in lysozyme M-/- mice compared with WT mice. The proinflammatory cytokine IL-12 was significantly elevated in lysozymetg mice and significantly decreased in lysozyme M-/- mice compared with WT mice.



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  		Figure 5. Levels of cytokines in lung homogenates after intratracheal instillation of K. pneumoniae. Lysozyme M-/- (black bars), lysozymetg (hatched bars), and WT (white bars) mice were infected with 104 CFU of K. pneumoniae. Lungs were harvested and homogenized at 24 hours after infection. Concentrations of tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin (IL)-6, IL-10, IL-12, macrophage inflammatory protein-2 (MIP-2), and released upon activation normal T cell expressed and secreted were quantitated by ELISA (n = 5 mice for each group). *p < 0.02 for WT versus lysozyme M-/-; **p < 0.04 (IL-10) and 0.01 (IL-12) for WT versus lysozymetg.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study assessed the role of the major form of lysozyme in the lung (lysozyme M) in killing an important human airway pathogen, K. pneumoniae. Increased expression of lysozyme in the airspaces of transgenic mice reduced bacterial burden and enhanced survival after intratracheal infection with K. pneumoniae. Targeted deletion of the lysozyme M gene resulted in decreased killing of K. pneumoniae and significantly increased mortality. These results suggest that lysozyme plays an important role in protecting the airways against infection by K. pneumoniae.

Deficiency of lysozyme M was associated with colonization of lung tissues with Gram-positive, catalase-negative bacilli bacteria that were identified morphologically as lactobacilli. Lactobacilli are lactic acid–producing bacteria, comprised of at least 70 distinct species (12, 13). Bacteria of this genus are normal components of oropharyngeal, vaginal, and gastrointestinal microflora of healthy individuals, the predominant species being Lactobacillus plantarum and Lactobacillus rhamnosus. Lactobacilli are generally considered to be of low pathogenicity, and some species have received considerable attention as candidate probiotics (14); however, under certain circumstances, such as in immunocompromised patients, lactobacilli may become an opportunistic pathogen (15). Detection of lactobacilli in lung tissues of lysozyme M-/- mice, but not WT littermates housed in the same cage, suggests that the M isoform of lysozyme may play a role in limiting the spread of oropharyngeal microflora.

The lysozymetg mice used in this study were generated in the FVB/N genetic background, whereas the lysozyme M-/- mice were generated in the C57BL/6 background (3, 9). The results of preliminary experiments indicated that the lethal dose 50% for K. pneumoniae was higher for WT mice of the C57BL/6 strain than the FVB/N strain. For this reason, lysozyme M-/- mice were backcrossed through 10 generations to FVB/N mice. When challenged with 105 CFUs of K. pneumoniae, all lysozyme M-/- mice died by 72 hours; similarly, when lysozyme M-/- mice in the C57BL/6 background were challenged, all mice died by 84 hours after infection (data not shown). In contrast to these results, 25% and 75% of WT and lysozymetg mice, respectively, survived to 120 hours. These results clearly indicate that elevated levels of lysozyme confer resistance to morbidity and mortality associated with K. pneumoniae infection.

Targeted inactivation of the lysozyme M gene resulted in an unexpected increase in lysozyme P expression (6) (Markart and colleagues, unpublished results). Despite partial compensation by P lysozyme, muramidase activity in BALF from lysozyme M-/- mice was significantly lower than that in WT mice. Consistent with reduced muramidase activity, bacterial killing was significantly decreased in lysozyme M-/- mice after intratracheal infection with K. pneumoniae (this study). The observation that some muramidase activity was detected in lysozyme M-/- mice suggested that targeted deletion of both the M and P loci would further increase the morbidity and mortality associated with infection by K. pneumoniae.

Lysozyme may act through multiple pathways to enhance bacterial killing. Both lysozyme M and lysozyme P directly kill K. pneumonia in vitro (Markart and colleagues, unpublished results). The bactericidal properties of lysozyme are likely mediated through enzymatic (muramidase) as well as nonenzymatic mechanisms (e.g., lytic peptide domains) (16–19). Lysozyme deficiency leads to increased bacterial load associated with persistent inflammation, which, in turn, may lead to deranged cytokine profiles. Intratracheal infection of mice with K. pneumoniae was previously reported to be associated with increased concentration of the antiinflammatory cytokine IL-10 (20, 21). Passive immunization of mice with anti–IL-10 serum before administration of bacteria resulted in decreased bacterial burden and increased survival associated with enhanced expression of proinflammatory cytokines (22). In particular, the proinflammatory cytokine IL-12 was shown to be an important component of host defense against K. pneumoniae (23). Adenoviral-mediated expression of IL-12 in the lung resulted in increased survival, whereas inhibition of IL-12 bioactivity resulted in decreased bacterial clearance and survival of infected mice. Consistent with these reports, enhanced survival of lysozymetg mice was associated with increased concentration of IL-12 and decreased concentration of IL-10 in lung homogenates after intratracheal infection with K. pneumoniae; in contrast, IL-10 concentration was increased, and IL-12 concentration decreased in lung homogenates of lysozyme M-/- mice. Although these data are consistent with the known roles of IL-12 and IL-10, it is not clear to what extent these relatively modest changes in cytokine levels contributed to the morbidity and mortality of infected mice in this study.

In summary, increased levels of lysozyme were protective, whereas deficiency of lysozyme M was associated with a worse outcome after acute airway infection with K. pneumoniae. These data support an important role for lysozyme in innate immunity and suggest that exogenously administered lysozyme may be a useful adjunct in the treatment of pneumonia due to K. pneumoniae.


   Acknowledgments
 
The authors thank Ann Maher for secretarial assistance and Dr. Thomas Graf for Lysozyme M-/- mice.


   FOOTNOTES
 
Supported by National Institutes of Health grant R01AI50797.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: P.M. has no declared conflict of interest; T.R.K. has no declared conflict of interest; T.E.W. has no declared conflict of interest; H.T.A. has no declared conflict of interest.

Received in original form May 16, 2003; accepted in final form November 8, 2003


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2004 American Thoracic Society