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Journal of Virology, February 2009, p. 1341-1349, Vol. 83, No. 3
0022-538X/09/$08.00+0     doi:10.1128/JVI.01123-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Infection with Human Metapneumovirus Predisposes Mice to Severe Pneumococcal Pneumonia

Irena Kukavica-Ibrulj,¹ Marie-Ève Hamelin,¹ Gregory A. Prince,² Constance Gagnon,¹ Yves Bergeron,¹ Michel G. Bergeron,¹ and Guy Boivin^1*

Research Center in Infectious Diseases of the Centre Hospitalier Universitaire de Québec and Laval University, Quebec City, Quebec, Canada,¹ Virion Systems, Inc., Rockville, Maryland²

Received 28 May 2008/ Accepted 2 November 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Human metapneumovirus (hMPV) is a recently described paramyxovirus that causes respiratory tract infections. Prior clinical studies have highlighted the importance of respiratory viruses, such as influenza virus, in facilitating secondary bacterial infections and increasing host immunopathology. The objective of the present work was to evaluate the effects of initial viral infection with hMPV or influenza A virus followed by Streptococcus pneumoniae superinfection 5 days later in a murine model. Both groups of superinfected mice demonstrated significant weight loss (mean of 15%) and higher levels of airway obstruction (mean enhanced pause value of 2.7) compared to those of mice infected with hMPV, influenza virus, or pneumococcus alone. Bacterial counts increased from 5 x 10² CFU/lung in mice infected with pneumococcus only to 10⁷ and 10⁹ CFU/lung in mice with prior infections with hMPV and influenza A virus, respectively. A more pronounced interstitial and alveolar inflammation correlated with higher levels of inflammatory cytokines and chemokines such as interleukin-1 (IL-1), IL-1β, IL-6, IL-12, monocyte chemotactic protein 1, macrophage inflammatory protein 1, KC, and granulocyte colony-stimulating factor, as well as greater expression of Toll-like receptor 2 (TLR2), TLR6, TLR7, and TLR13 in the lungs of superinfected animals compared to results for single infections, with similar immunological effects seen in both coinfection models. Prior infection with either hMPV or influenza A virus predisposes mice to severe pneumococcus infection.

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Human metapneumovirus (hMPV) is a newly discovered member of the Metapneumovirus genus within the Paramyxoviridae family (46). This virus has been associated with various upper and lower respiratory tract syndromes, including common colds, bronchitis, pneumonia, and asthma exacerbation, with more severe diseases reported for young children, elderly subjects, and immunocompromised patients (6, 9, 18, 22, 38, 46). Several studies have shown that hMPV is a worldwide pathogen that infects virtually all individuals by the age of 5 years and that results in a significant percentage of hospitalizations of young children (7, 15, 46, 49).

Data on the role of coinfecting pathogens in hMPV-infected individuals are limited and have focused almost exclusively on the interaction between hMPV and other respiratory viruses, in particular the potential synergistic association with another paramyxovirus, i.e., the human respiratory syncytial virus (hRSV) (14, 17, 23, 24, 30, 40, 48). Bacterial coinfections previously have been reported by our group for hMPV-infected individuals (6). Two of 12 (16.7%) hMPV-infected hospitalized children from Canada harbored Streptococcus pneumoniae (pneumococcus) or Staphylococcus aureus in their respiratory tract specimens. Additionally, it has been suggested that the pathogenesis of severe hMPV-associated lower respiratory tract infections in South African children involves coinfection with pneumococcus, since vaccination with a 9-valent pneumococcal conjugate vaccine prevented hMPV-related hospitalizations (27). Similarly, pulmonary bacterial coinfections have been reported for children with severe hRSV bronchiolitis (45), and recent hospitalization for hRSV infection has been shown to increase the risk of invasive pneumococcal disease (42).

The synergistic interaction between the influenza A virus (a member of the Orthomyxoviridae family) and pneumococcus is well documented in humans (31) and has been extensively studied in animal models (39, 41). We used a well-established experimental murine model to validate our hypothesis that hMPV, like influenza virus, increases pneumococcus replication in lungs and enhances host immunological responses.

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Cell lines and viruses. Rhesus monkey kidney (LLC-MK2), Madin-Darby canine kidney (MDCK), and Madin-Darby bovine kidney (MDBK) cells were maintained in minimal essential medium (MEM; Gibco, Invitrogen, Burlington, Ontario, Canada) supplemented with 10% fetal bovine serum. The hMPV clinical strain C-85473 (group A) (20) and the influenza A/WSN/33 (H1N1) recombinant virus (2) were used in this study.

Virus propagation and quantification. hMPV was grown in LLC-MK2 cells and quantified using OptiMem I medium (Gibco) supplemented with 2 µg/ml of trypsin (Sigma, Oakville, Ontario, Canada) and GVF antibiotic (1 µg/ml of gentamicin, 25 µg/ml of vancomycin, and 0.125 µg/ml of amphotericin B). When indicated, hMPV inactivation was achieved by heating the viral preparation for 30 min at 56°C. hMPV titers in lung homogenates were determined as previously described (20) and were reported as 50% tissue culture infective doses (TCID₅₀)/lung. Influenza virus initially was amplified in MDCK cells and quantified on confluent monolayers of MDBK cells using Eagle's MEM supplemented with 0.5% bovine serum albumin fraction V and GVF. Influenza virus titers were determined as previously described (1) and are reported as PFU/lung.

Pneumococcus propagation and quantification. A penicillin-susceptible Streptococcus pneumoniae serotype 3 clinical isolate was used in this study. Bacteria were grown in brain heart infusion broth to mid-logarithmic phase at 37°C as previously described (16). The bacterial counts then were confirmed by duplicate plating on blood agar. Aliquots (100 µl) of lung homogenates were collected from animals, serially diluted, and plated in duplicates onto blood agar. The plates were incubated for 16 to 18 h at 37°C in 5% CO₂. The number of colonies was manually counted, and bacterial titers were expressed as log₁₀ CFU/lung.

Mouse model of viral-bacterial infections. Female BALB/c mice (4 to 6 weeks of age) were obtained from Charles Rivers Laboratories (Wilmington, MA) and were housed in sterile microisolators. All procedures were approved by the Laval University Institutional Animal Protection Care and Use Committee.

Mice were infected intranasally on day 0 with low viral inocula, i.e., inocula not producing overt clinical symptoms (1.5 x 10⁵ TCID₅₀ of hMPV or 10² PFU of influenza virus) followed, in some cases, by a superinfection 5 days later with 10³ CFU of S. pneumoniae. For the hMPV-pneumococcus coinfection model, two additional experiments were performed: (i) challenge with heat-inactivated hMPV followed by a pneumococcus superinfection 5 days later, and (ii) challenge with live hMPV followed by delayed pneumococcal challenge 14 days later. The experimental protocols included different groups of mice: group A received influenza virus only followed by phosphate-buffered saline (PBS); group B received influenza virus and then S. pneumoniae; group C received hMPV only followed by PBS; group D received hMPV and then S. pneumoniae; group E received PBS and then S. pneumoniae; and group F received PBS twice (sham infection). In addition, group G received heat-inactivated hMPV followed by S. pneumoniae 5 days later, while group H received live hMPV followed by delayed S. pneumoniae infection 14 days later (at a time when there is no more replicating virus). Mice were monitored daily for overt disease symptoms (ruffled fur, inactivity, weight loss, and the presence of any respiratory symptoms). Mice were sacrificed when they had lost >20% of their initial weight or at the end of the experiment.

Each group of mice included 18 animals (except for groups G and H, which had 6 animals each), with 6 mice sacrificed on days 6, 7, and 8 (day 8 only for group G and day 17 only for group H). Days 6, 7, and 8 correspond to 24, 48, and 72 h after bacterial infection or PBS treatment. Four experimental parameters were evaluated in this study. First, the level of airway obstruction was evaluated by measuring breathing patterns using a plethysmograph. Bacterial and viral loads, the degree and type of inflammation, and overall host immune responses then were evaluated in the lungs of sacrificed mice.

Airway obstruction. Breathing patterns were characterized in groups of six mice at each time point using an unrestrained whole-body flowthrough plethysmograph system as previously reported (19). Mice were allowed to acclimate to the chamber for several minutes, respiratory parameters were recorded for 5 min, and then enhanced pause (P_enh) values were calculated for determining airways obstruction (25).

Viral and bacterial lung titration studies. Six mice per group were sacrificed at each time point, and then lungs were excised and homogenized in 1 ml of sterile PBS for the determination of viral or bacterial loads. Serial dilutions of homogenized lungs were laid on LLC-MK2 monolayers for hMPV and on MDBK monolayers for influenza virus titration studies, as reported above, with the addition of 0.5 µg/ml of penicillin G for S. pneumoniae-superinfected groups. For bacterial quantification, aliquots of lung homogenates were serially diluted and plated onto blood agar as described above.

Pulmonary histopathology. Lungs from six mice per group were removed at each time point and fixed with 3.7% buffered formaldehyde. Fixed lungs were embedded in paraffin, sectioned in 5-µm slices, and stained with hematoxylin-eosin. The histopathologic score was determined by a pathologist unaware of the infection status of the animals. Four types of histopathological changes were scored independently (peribronchiolitis, perivasculitis, interstitial pneumonitis, and alveolitis) and were evaluated to determine histopathological scores based on a scale ranging from 0 to 4 as previously described (20, 37).

Pulmonary cytokine/chemokine levels. Aliquots of 750 µl from the same lung homogenates used for viral and bacterial titration studies were mixed with 750 µl of potassium buffer containing 0.2% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]1-1-propanesulfonate} and 0.2% of a protease inhibitor cocktail (Sigma, Oakville, Ontario, Canada). Samples were centrifuged at 13,800 x g for 10 min at 4°C, and the supernatants were stored at –20°C for pulmonary cytokine/chemokine quantification. The simultaneous evaluation of multiple cytokines/chemokines was performed using a Luminex 100 instrument system (Qiagen, Mississauga, Ontario, Canada) and the Bio-Plex mouse cytokine 23-plex panel (Bio-Rad Laboratories, Mississauga, Ontario, Canada). This allows the quantification of 23 different cytokines/chemokines, i.e., interleukin-1 (IL-1), IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, granulocyte-monocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), tumor necrosis factor alpha (TNF-), gamma interferon (IFN-), the mouse IL-8 homologue KC, monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 (MIP-1), MIP-1β, eotaxin, and RANTES. The assay was performed using 50 µl of lung homogenate as specified by the manufacturer.

Quantitative real-time PCR analysis for pulmonary TLR expression. Six mice per group were sacrificed at each time point, and then lungs were excised and homogenized in 1 ml of TRIzol reagent and rapidly frozen on dry ice for the subsequent determination of pulmonary toll-like receptor (TLR) expression levels. Total RNA extraction was performed using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol and then digested with DNase (Turbo DNA-free; Ambion, Austin, TX). RNA quantity and quality was assessed using an Agilent Technologies 2100 bioanalyzer and the RNA 6000 Nano LabChip kit (Agilent, Mountain View, CA). cDNA was generated from 5 µg of total RNA using a random primer hexamer and the Superscript II reverse transcriptase (Invitrogen). Equal amounts of cDNA were run in duplicate and amplified in a 15-µl reaction mix containing 7.5 µl of 2x universal PCR master mix (Applied Biosystems, Foster City, CA), 10 nM of Z-tailed forward primer, 100 nM of reverse primer, 100 nM of Amplifluor UniPrimer probe (Chemicon, Temecula, CA), and 2 µl of target DNA. The mixture was incubated at 50°C for 2 min and at 95°C for 4 min, and then it was cycled 55 times at 95°C for 15 s and at 55°C for 40 s using the Applied Biosystems Prism 7900 sequence detector. Results for the amplification products were normalized to values for the 18S ribosomal gene. Primers were designed using Primer Express 2.0 (Applied Biosystems), and their sequences are available upon request. Amplicons were detected using the Amplifluor UniPrimer system in which forward primers contained the 5' Z sequence ACTGAACCTGACCGTACA.

Statistical analysis. Statistical analyses were performed with the GraphPad Prism 5 software using unpaired t tests or nonparametric Mann-Whitney tests.

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Clinical signs of mice with single and dual infections. To determine whether the synergistic exacerbation of disease could be seen in a mouse model of dual virus-bacterium infections, animals first were infected with low inocula of hMPV or influenza A virus that did not result in any clinical signs or weight loss and subsequently infected, 5 days later, with a nonlethal dose of S. pneumoniae. Initial infections with either virus followed by bacterial superinfection resulted in severe and rapid weight loss (mean of 15% on day 8) associated with breathing difficulties, ruffled fur, and inactivity (Fig. 1). Three days after bacterial superinfection, a mortality rate of 100% was observed in both coinfection models (data not shown). In contrast, no clinical signs or weight loss were observed when heat-inactivated hMPV infection was followed by S. pneumoniae coinfection 5 days later or when S. pneumoniae infection was delayed 14 days after live hMPV infection (data not shown).

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FIG. 1. Weight loss in groups of mice with viral, bacterial, or dual infection. Groups include mice with hMPV-only infection (), hMPV-pneumococcus coinfection (), influenza A virus-only (Flu) infection (), influenza A virus-pneumococcus coinfection (•), pneumococcus-only infection (), and sham (PBS) infection (). *, P 0.05 for the comparison between single infections and coinfections (unpaired t test).

Airway obstruction in mice with single and dual infections. As shown in Fig. 2, the degree of airway obstruction (reported as P_enh values) in coinfected mice was significantly higher than that of mice infected with a single virus only or sham-infected mice. This effect was seen on days 7 and 8 in hMPV-pneumococcus-infected mice and on days 6, 7, and 8 in influenza virus-pneumococcus-infected mice.

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FIG. 2. Airway obstruction in groups of mice with viral, bacterial, or dual infection. Six mice/group were evaluated at different time points (days 6, 7, and 8 after virus infection, corresponding to 24, 48, and 72 h following bacterial superinfection, respectively) after sham infection, single infections, and virus-bacterium coinfections. Flu, influenza virus. Airway obstruction (reported as Penh values) was determined using whole-body unrestrained plethysmography. P 0.05 for the comparison to results for sham-infected mice (#) and to virus-only infections (*) (unpaired t test).

Viral and bacterial titers in the lungs. There were no significant differences in pulmonary virus titers between groups of mice with single viral infections and those with coinfections (Fig. 3A). As expected, a gradual decrease in lung viral titers was observed over time in mice with single viral infections and in those with coinfections. In addition, hMPV replication was not found after challenge with the heat-inactivated virus or at the time of delayed pneumococcal challenge on day 14 (corresponding to day 17 after hMPV infection) (data not shown).

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FIG. 3. Viral and bacterial loads in groups of mice with viral, bacterial, or dual infection. Six mice per group were infected with either hMPV, influenza virus (Flu), or PBS (sham), challenged 5 days later with pneumococcus or PBS, and then sacrificed for quantitative viral (A) or bacterial (B) pulmonary titers on days 6, 7, and 8 after virus infection (corresponding to 24, 48, and 72 h following bacterial superinfection, respectively). *, P 0.05 for the comparison between single infections and coinfections (Mann-Whitney test).

In sharp contrast, significantly higher pulmonary bacterial counts were observed in mice with previous viral infections compared to those with bacterial infection only (Fig. 3B). The increase in bacterial counts on day 8 was 4 log₁₀ for hMPV-pneumococcus-infected and 6 log₁₀ for influenza virus-pneumococcus infected mice. In the experiment with the heat-inactivated hMPV followed by pneumococcus infection 5 days later, we did not see any significant increase in the pulmonary pneumococcus counts on day 8 (P = 0.095). Similarly, when pneumococcus challenge was delayed 14 days after the initial hMPV infection, the difference between the hMPV-pneumococcus group and the pneumococcus-only group was not significant on day 17 (P = 0.15).

Lung histopathology in mice with single and dual infections. Microscopic examination of hematoxylin and eosin-stained lung sections showed the presence of some inflammation in hMPV- or influenza virus-infected mice that gradually decreased from days 6 to 8 (Fig. 4A). The lungs of pneumococcus- and sham-infected mice had relatively comparable levels of inflammation. In contrast, lungs of superinfected mice showed pronounced inflammation and congestion that increased over time.

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FIG. 4. Lung histopathological evaluation of mice with viral, bacterial, or dual infection. Six mice per group were euthanized at different time points after infection, and their lungs were removed and fixed with 3.7% buffered formalin, cut, and stained with hematoxylin and eosin. (A) Representative sections (magnification, x10) are shown for days 6, 7, and 8 after virus infection (corresponding to 24, 48, and 72 h after bacterial superinfection, respectively) for mice with sham, single, or dual infection. Flu, influenza virus. (B) Corresponding histopathological scores also are reported for the same groups of mice. The degree of lung inflammation (mean histopathological score) was evaluated for peribronchial, perivascular, interstitial, and alveolar areas.

As shown in Fig. 4B, the inflammatory process seen in virus-only and both coinfection models mostly consisted of interstitial inflammation (characterized by the increased thickness of the alveolar walls) and alveolitis (characterized by the presence of inflammatory cells within alveolar spaces). The total histopathological scores (i.e., the sum of all four types of inflammation evaluated) were similar for mice with single infections and coinfections on day 6 (i.e., 8.8, 8.4, 9.5, and 8.7 for hMPV only, hMPV-pneumococcus, influenza virus only, and influenza virus-pneumococcus). Histopathological scores were increased on day 8 in mice with superinfections compared to those of mice with single infections (i.e., 10, 11, 8.6, and 11 for hMPV only, hMPV-pneumococcus, influenza virus only, and influenza virus-pneumococcus), and this was due mainly to increased interstitial and alveolar inflammation.

Cytokine and chemokine pulmonary levels in mice with single and dual infections. To further understand the pathogenesis of sequential coinfections with respiratory viruses and S. pneumoniae, a broad array of cytokines and chemokines was measured in the lungs of infected mice on days 6, 7, and 8 postinfection. Among the 23 cytokines and chemokines evaluated, none were significantly increased in S. pneumoniae-infected mice compared to the levels in sham-infected mice (data not shown), which reflects the low bacterial inoculum used in these experiments. In contrast, IL-1, IL-1β, IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, GM-CSF, KC, MCP-1, MIP-1, MIP-1β, and RANTES were significantly increased in hMPV- and influenza virus-infected mice compared to the levels in sham-infected mice (data not shown). Compared to levels for virus-only infections, the levels of 13 cytokines/chemokines were significantly increased after coinfection with hMPV and pneumococcus (Fig. 5A), whereas 15 were significantly increased after coinfection with influenza virus and pneumococcus (Fig. 5B).

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FIG. 5. Cytokine and chemokine levels in the lungs of mice with single-virus-only or dual viral-bacterial infections. Six mice per group were euthanized at different time points after infection (on days 6, 7, and 8 after virus infection, corresponding to 24, 48, and 72 h following bacterial superinfection, respectively) for the evaluation of 23 pulmonary cytokines and chemokines by the use of a Luminex 100 instrument system and the Bio-Plex mouse cytokine 23-plex panel. Values are expressed as changes (n-fold) in pulmonary levels for mice with coinfections compared to those of mice with hMPV-only infection (A) or influenza A virus-only (Flu) infection (B). *, P 0.05 for the comparison between single infections and coinfections (unpaired t test).

Similar cytokine/chemokine profiles were observed in both coinfection models, including significantly increased levels of the proinflammatory cytokines TNF-, IFN-, IL-1, IL-1β, IL-6, and IL-12 (p70) as well as the antiinflammatory cytokine IL-13 and the chemokines MIP-1, MCP-1, and KC. Of particular note is the very important increase of G-CSF and, to a lesser extent, GM-CSF in lungs of superinfected animals.

Coinfection with influenza virus and pneumococcus differed from that of hMPV and pneumococcus by increased levels of IL-10 and IL-12 (p40).

Pulmonary expression of TLR in mice with single and dual infections. All known mouse TLR transcripts were quantified in the lungs of infected mice on days 6, 7, and 8 after virus infections using real-time PCR. The expression levels of all pulmonary TLRs were similar for mice with pneumococcus-only and sham infections (data not shown). There was an increased expression of TLR1, TLR9, TLR11, TLR12, and TLR13 for hMPV-infected mice and TLR1, TLR9, and TLR13 for influenza virus-infected mice compared to the levels for sham-infected mice (data not shown). Figure 6 shows the changes (n-fold) in pulmonary TLR transcripts for mice with coinfections compared to those with single-virus-only infections. In both coinfection models, there was an increased expression of TLR2, TLR6, TLR7, and TLR13 compared to that of single infections on days 7 and 8 postinfection. In addition, TLR3, TLR4, and TLR8 transcripts were significantly increased in the influenza virus-pneumococcus coinfection model, whereas the level of TLR1 was significantly elevated on day 7 in the hMPV-pneumococcus coinfection model. Interestingly, the expression levels of TLR11 and TLR12 were significantly decreased on day 8 in mice with hMPV-pneumococcus coinfection compared to those in mice with hMPV infection only.

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FIG. 6. TLR transcript levels in the lungs of mice with single-virus-only or dual viral-bacterial infections. RNA was isolated from the lung tissues of infected animals on days 6, 7, and 8 after virus infections (corresponding to 24, 48, and 72 h following bacterial superinfection, respectively) for TLR quantification using real-time PCR. Values represent changes (n-fold) in pulmonary expression levels for mice with coinfections compared to those of mice with single infection with hMPV (A) or with influenza A virus (Flu) (B). *, P 0.05 for the comparison between single infections and coinfections (unpaired t test).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we investigated the synergistic effect of hMPV and pneumococcus infections in a mouse model. In order to validate our results, we used a well-established influenza virus-pneumococcus synergistic coinfection model as a control. Our results demonstrate that hMPV, like influenza A virus, predisposes animals to severe bacterial infections. The high bacterial loads observed in the lungs of mice following sequential viral-bacterial infections and the release of several proinflammatory cytokines may have both contributed to the adverse clinical outcome. The in vivo studies described here using sequential low viral and bacterial inocula are highly reminiscent of the synergistic disease observed in the clinical settings, and such a model could be used to evaluate the benefits of antiviral, antibiotic, vaccine, and immunomodulatory approaches in decreasing morbidity and mortality.

Secondary bacterial infections often complicate respiratory viral infections, although the mechanisms whereby viruses predispose the host to exacerbated bacterial disease are not completely understood. The most frequently postulated mechanisms include (i) virus destruction of the respiratory epithelium, which may increase bacterial adhesion; (ii) virus-induced immunosuppression, which can lead to bacterial superinfections; and (iii) an inflammatory response to viral infection that may upregulate the expression of molecules that bacteria utilize as receptors (34). Previous studies demonstrated that S. pneumoniae binds more efficiently to influenza virus-infected epithelial cells in vitro and in a mouse model (29, 36). This increased binding is believed to be the result of the complete desquamation of the respiratory epithelium following viral infection. Influenza and parainfluenza viruses possess a neuraminidase (NA), which appears to further increase bacterial adherence after viral preincubation. Indeed, experimental studies demonstrated that viral NA exposes pneumococcal receptors on host cells by removing terminal sialic acids (28, 29). The inhibition of viral NA activity using commercially available NA inhibitors has been shown to reduce the adherence and invasion of S. pneumoniae independently of its effects on viral replication (28). Several clinical studies also have revealed that influenza virus vaccination reduces the incidence of secondary bacterial respiratory tract infections (34). On the other hand, hMPV does not possess an NA protein, and different mechanisms may be involved to explain the synergistic interaction with S. pneumoniae. Indeed, it has been suggested that hMPV-associated lower respiratory tract infections resulting in hospitalization frequently involve coinfection with pneumococcus, since a significant proportion of these hospitalizations could be prevented by vaccination with the pneumococcal conjugate vaccine (27). Furthermore, a recent in vitro study has confirmed that other paramyxoviruses (hRSV and parainfluenza virus 3) could increase bacterial adherence to primary bronchial epithelial cells and immortalized cell lines by upregulating eukaryotic cell receptors for nontypeable Haemophilus influenzae and S. pneumoniae, whereas this mechanism was less significant in primary small airway epithelial cells and in influenza virus infections (4). Our study demonstrated that challenge with inactivated hMPV did not enhance clinical symptoms or pneumococcal replication in the lung, suggesting that active hMPV replication is required for exacerbated pneumococcal disease. Delayed pneumococcus superinfection 14 days after hMPV infection led to similar results, confirming that the enhancing effect of pneumococcus is driven primarily by replicating hMPV and much less by a long-lasting associated host response to hMPV, which contrasts with the response to influenza virus (41). Thus, our results show that despite similar clinical outcomes, hMPV and influenza virus use different mechanisms to enhance pneumococcus replication in the lung. hMPV may use the same mechanism as the other paramyxoviruses in promoting bacterial adherence, since our group showed that it affects the same bronchial epithelial cells based on lung autopsy materials from a fatal hMPV case (8). Additional studies are needed to understand the different mechanisms underlying the synergistic interaction between hMPV and bacteria.

An excessive host immune response and sustained inflammation following viral infection can be a factor responsible for increased susceptibility to secondary bacterial pneumonia. During pulmonary infections, the host response involves the coordinated expression of early response cytokines (e.g., TNF-, IL-1β, and IL-6) by alveolar macrophages, dendritic cells, and other antigen-presenting cells; the release of chemotactic cytokines for the recruitment of neutrophils (e.g., IL-8, KC, and MIP-2), macrophages (e.g., MIP-1), monocytes (e.g., MCP-1), and lymphocytes; and the participation of activating/inhibiting cytokines (e.g., IFN- from Th-1 lymphocytes and IL-10 from Th-2 lymphocytes) for the resolution of infection and long-term protection against reinfection (5). In our study, very important levels for a variety of proinflammatory molecules were observed in the lungs of superinfected animals, including TNF-, IFN-, IL-1, IL-1β, IL-6, and IL-12 (p70). These cytokines can lead to local inflammation and tissue damage when produced in excess amounts. Thus, our results suggest that multiple inflammatory mediators are synergistically exacerbated and contribute to the morbidity and mortality seen in our coinfection models. Additionally, increased pulmonary levels of IL-9, IL-10 (for influenza virus-pneumococcus coinfection only), and IL-13 were also demonstrated in our study. IL-9, a Th2-type cytokine, has been suggested as a candidate factor for asthma (10, 12, 33, 44). IL-10, an antiinflammatory cytokine, is an important mediator of enhanced pneumococcal pneumonia after influenza virus (47). Furthermore, it has been demonstrated that hRSV infection can induce IL-13-dependent changes in airway function and promote an environment that contributes to the development of severe allergic asthmatic responses (26). Airway hyperresponsiveness has been associated with hMPV infections in humans (22) and in animal models (19). The increased expression of the latter cytokines (IL-9, -10, and -13) could explain the significant airways obstruction seen in our virus-bacteria coinfection models and also in humans.

High levels of MIP-1, MCP-1, and the neutrophil chemoattractant KC (the mouse homologue of human IL-8) have been found in the lungs of superinfected animals. Upon activation, neutrophils produce proinflammatory cytokines, which cause fever and local inflammation, and also help in the recruitment of additional inflammatory cells, including T and B lymphocytes (13). Activated neutrophils also produce antimicrobial agents, such as hydrogen peroxide, superoxide anion, and nitric oxide (13). Thus, the enhanced bacterial colonization of the lungs following virus infection may lead to the sustained production of toxic compounds by neutrophils, resulting in tissue destruction (41). In addition, levels of G-CSF, which regulates the maturation, differentiation, and proliferation of neutrophils, were highly elevated in the lungs of superinfected animals. Thus, the high pulmonary levels of proinflammatory cytokines and G-CSF produced by the inflamed endothelium, alveolar monocytes, and macrophages likely contributed to the enhanced pneumococcal disease seen after hMPV or influenza virus infection.

The production of cytokines and chemokines in response to components of microbiological agents is stimulated by TLRs and mediated by mitogen-activated protein kinase signaling (3, 35). TLR2, TLR6, TLR7, and TLR13 were specifically expressed in lungs of mice in both coinfection models. In addition, influenza virus-S. pneumoniae coinfection induced the expression of TLR3 and TLR4, especially at 72 h following pneumococcal superinfection. TLR2 is known as the recognition receptor for peptidoglycans of gram-positive bacteria, including S. pneumoniae (50). The recognition of microbial components by TLR2 requires cooperation with other TLRs. The coexpression of TLR2 and TLR6 at the cell surface is crucial for the recognition of diacylated lipopeptide and peptidoglycan and subsequent cellular activation in human cells (32). It has been shown that TLR7/TLR8 agonists can stimulate Th1-type cellular responses with the production of IFN- and other cytokines such as TNF- and IL-12 from rat peripheral blood mononuclear cells in order to inhibit influenza virus infection (21). Interestingly, pulmonary levels of TLR11 and TLR12 were lower in coinfected mice than in animals with hMPV infection only. These data suggest that TLR11 and TLR12 are implicated in the recognition of specific hMPV components. TLR11, TLR12, and TLR13 recently have been discovered and lack human counterparts (43). Additional studies are required to understand the role of TLR13, which was the most significantly expressed TLR in both coinfection models. Our results contrast with those of Didierlaurent et al., suggesting that the bacterial desensitization of TLRs following influenza virus infection is associated with reduced chemokine production and neutrophil recruitment (11). Differences in viral strains, doses, and superinfection schedule could explain this discrepancy.

Clearly evident from our study and previously confirmed by other groups for influenza virus (39, 41) is the concept that a viral infection (with hMPV or influenza virus) creates an environment in the respiratory tract that predisposes the host to an exacerbated response to subsequent bacterial infections. Indeed, the low viral inocula that we used for initial infection resulted in a minimal increase of proinflammatory cytokines (with the exception of that for IL-12 [p40]) compared to those of sham-infected mice, whereas superinfected mice displayed a dramatic increase in pulmonary levels of the same cytokines. This suggests that activated lymphocytes/monocytes were predisposed to secrete massive levels of chemokines and proinflammatory cytokines once the superinfection occurred.

In summary, we have established a murine model that closely mimics the clinical exacerbation of hMPV-associated respiratory disease by S. pneumoniae. The use of subclinical doses of each organism was sufficient to induce a severe and lethal pulmonary disease. Coinfection with hMPV and pneumococcus, similarly to coinfection with influenza virus and pneumococcus, results in the massive expression of neutrophil chemoattractants and activators, such as MCP-1, MIP-1, KC, and especially G-CSF. G-CSF may increase neutrophil survival, allowing the sustained secretion of tissue-damaging molecules in the lungs of superinfected animals (41). Although hMPV and influenza virus can predispose the host to severe pneumococcal pneumonia through different mechanisms, the outcomes were similar in both cases. Our work underlines the need for designing effective anti-hMPV compounds with an emphasis on the development of prophylactic modalities, i.e., monoclonal antibodies and vaccines for this important viral pathogen. In addition, our results stress the importance of the conjugate pneumococcal vaccine in reducing the severity of hMPV and influenza virus infections by preventing pneumococcal superinfections.

 
This study was supported by research grants from the Canadian Institutes of Health Research (MOP-62789) to G.B. G.B. is a national scholar of the FRSQ and the holder of the Canada Research Chair on Emerging Viruses and Antiviral Resistance. I.K-I. is a postdoctoral scholar from the FRSQ-Respiratory Health Network.

We acknowledge Jacques Corbeil for quantitative real-time PCR analysis and the contribution of the Gene Quantification Core Laboratory of the Centre de Génomique de Québec.

We have no conflicting financial interests.

 
* Corresponding author. Mailing address: CHUQ-CHUL, room RC-709, 2705 Blvd. Laurier, Quebec City, Quebec, Canada G1V 4G2. Phone: (418) 654-2705. Fax: (418) 654-2715. E-mail: Guy.Boivin{at}crchul.ulaval.ca

Published ahead of print on 19 November 2008.

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Journal of Virology, February 2009, p. 1341-1349, Vol. 83, No. 3
0022-538X/09/$08.00+0     doi:10.1128/JVI.01123-08
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