Decreased pulmonary clearance of S. pneumoniae following influenza A infection in mice

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Abstract

In children, the incidence of complicated pneumonias (including empyemas and lung abscesses) associated with Streptococcus pneumoniae infection has increased in recent years. In many cases, these complicated pneumonias followed flu-like illnesses. To determine mechanisms behind this association, a murine model of sequential pulmonary infection has been developed. BALB/cJ mice infected with influenza A had mild pulmonary inflammation that resolved within 5–7 days. Seven days following their initial ‘treatment’ (mock infection or influenza exposure), mice were challenged with 106 cfu of S. pneumoniae, and their lungs were harvested at intervals for analysis. Lungs of influenza-exposed mice demonstrated greater colony counts 24 and 48 h following S. pneumoniae exposure compared to control mice. In addition, neutrophil numbers were significantly increased in the influenza/S. pneumoniae sequentially-infected animals compared to S. pneumoniae infection alone (1.4±0.6×106 vs. 0.06±0.07×106 cells, P<0.05, 24 h). Influenza-exposed animals had greater levels of IL-1β and TNF-α in lung homogenates following S. pneumoniae inoculation. These data demonstrate that mice exposed to influenza have enhanced inflammatory responses and increased bacterial burden following S. pneumoniae exposure than do control mice. This model will be useful in defining mechanisms behind the enhanced susceptibility to S. pneumoniae that occurs after influenza exposure.

Introduction

In the USA, pneumonia and lower respiratory tract infections remain among the most common causes of hospitalizations in the pediatric population. The highest incidence of pneumonia occurs in children between 6 months and 5 years of age (Glezen, 1983, Denny and Clyde Jr, 1986, Wright et al., 1989), but the highest frequency of hospitalization for pneumonia occurs in children <1 year of age (Glezen, 1983). The agents causing these episodes of pneumonia cannot be identified in a significant proportion of these children — only 20–50% of cases in large epidemiologic studies (Mufson et al., 1970, Glezen, 1983). In these studies, pneumonia was found to occur primarily during epidemics of respiratory syncytial virus (RSV), parainfluenza virus, or influenza infection (Glezen, 1983, Denny and Clyde Jr, 1986, Wright et al., 1989).

Influenza viruses infect the epithelial cells of the upper and lower respiratory tract. Histologic analysis of influenza infection in humans (Walsh et al., 1961) or animal models (Smith and Sweet, 1988, Piazza et al., 1991, Lu et al., 1999) demonstrate similar pathology: acute influenza infection of the lower respiratory tract leads to desquamation of the epithelium down to the basal cell layer, resulting in clinical tracheobronchitis and bronchiolitis.

Pneumonia complicating influenza infection may be the direct result of viral infection, bacterial superinfection, or the combined viral/bacterial infection (Ruben and Cate, 1987). During the three influenza pandemics in this century (1918, 1957, and 1968), a significant portion of the influenza-associated mortality resulted from concurrent bacterial infections (Streptococcus pneumoniae or S. aureus, and others) rather than the acute influenza respiratory infection itself. Bacterial superinfections of the lower respiratory tract occur mainly in people at either end of the age spectrum and in those with underlying illness, however, immunocompetent children and adults can also succumb to these secondary infections (Abramson and Wheeler, 1994). Studies in human volunteers demonstrate that influenza A infection enhances rates of colonization by S. pneumoniae (Wadowsky et al., 1995), potentially setting the stage for secondary bacterial infection.

An increased incidence of complicated pneumonias associated with S. pneumoniae infection has recently been observed in otherwise healthy children (Hardie et al., 1996, Rey Rosa et al., 1996, Hardie et al., 1998). Frequently, these complicated pneumonias were associated with a clinical history of a recent flu-like illness, and elevated antibody titers to influenza A were demonstrated in a significant portion of these patients (Rey Rosa et al., 1996). These observations suggest that an antecedent influenza infection predisposes the otherwise healthy patient to subsequent bacterial superinfection and more severe disease.

Although human, and animal studies suggest influenza virus predisposes the host to secondary bacterial infections, the mechanism of this enhanced pathology remains uncertain. The studies reported here demonstrate the effects of antecedent influenza A exposure on pulmonary inflammation and bacterial clearance during subsequent exposure to S. pneumoniae, providing a model that should be useful in unraveling the mechanism(s) of bacterial superinfection following influenza respiratory tract infections.

Section snippets

Reagents

Tissue culture media and reagents, including Hank's Balanced salt solution (HBSS), Eagle's minimal essential medium (MEM), trypsin (0.5%) and fetal calf serum (FCS) were obtained from GIBCO BRL (Grand Island, NY). Plasticware (pipettes, flasks, tissue culture dishes, Falcon™) were obtained from Fisher Scientific. Media for propagation of S. pneumoniae were obtained from GIBCO/BRL (Grand Island, NY). Chemicals for buffers (phosphate buffered saline) were obtained from Sigma (St Louis, MO).

Infectious agents

Mouse

Susceptibility to S. pneumoniae following influenza A infection

Using the sequential infection (superinfection) model described above, BALB/cJ mice were exposed to influenza A, then 7 days later, exposed to S. pneumoniae. The mice were sacrificed at intervals indicated in Fig. 1, and the lungs or spleens homogenized and plated for bacterial colony counts. Prior exposure to influenza significantly decreased bacterial ‘clearance’ from the lung at 24 and 48 h following infection with S. pneumoniae compared to mice exposed to vehicle alone (P<0.01 at both time

Discussion

These studies demonstrate that prior exposure to influenza A decreases clearance of a secondary S. pneumoniae exposure from the lungs of BALB/cJ mice. However, this decrease in ability to remove/kill the S. pneumoniae occurs despite an overall increase in the numbers of inflammatory cells in the lung (reflecting primarily neutrophil influx). Bronchoalveolar lavage neutrophils isolated from mice following sequential infection generate equal levels of superoxide in response to PMA stimulation as

Acknowledgements

This research was supported by NIH grant HL02505 (J.M. Stark), the American Lung Association and the Cystic Fibrosis Foundation (J.M. Stark). The authors thank Dr Peter Gartside for his assistance with statistical analysis and Tracy Hopkins for technical support.

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